Structure of an integrin with an αI domain,complement receptor type 4

We report the structure of an integrin with an αI domain, α_Xβ₂, the complement receptor type 4. It was earlier expected that a fixed orientation between the αI domain and the β‐propeller domain in which it is inserted would be required for allosteric signal transmission. However, the αI domain is highly flexible, enabling two βI domain conformational states to couple to three αI domain states, and greater accessibility for ligand recognition. Although α_Xβ₂ is bent similarly to integrins that lack αI domains, the terminal domains of the α‐ and β‐legs, calf‐2 and β‐tail, are oriented differently than in αI‐less integrins. Linkers extending to the transmembrane domains are unstructured. Previous mutations in the β₂‐tail domain support the importance of extension, rather than a deadbolt, in integrin activation. The locations of further activating mutations and antibody epitopes show the critical role of extension, and conversion from the closed to the open headpiece conformation, in integrin activation. Differences among 10 molecules in crystal lattices provide unprecedented information on interdomain flexibility important for modelling integrin extension and activation.     

28-nor-oleanane-type triterpene saponins from Camellia japonica and their inhibitory activity on LPS-induced NO production in macrophage RAW264.7 cells

Four new 28-nor-oleanane-type triterpene oligoglycosides, camellenodiol 3-O-β-D-galactopyranosyl(1→2)[β-D-xylopyranosyl(1→2)-β-D-galactopyranosyl(1→3)]-β-D-glucuronopyranoside (2), camellenodiol 3-O-4'-O-acetyl-β-D-galactopyranosyl(1→2)[β-D-xylopyranosyl(1→2)-β-D-galactopyranosyl(1→3)]-β-D-glucuronopyranoside (4), camellenodiol 3-O-(β-D-galactopyranosyl(1→2)[β-D-xylopyranosyl(1→2)-β-D-galactopyranosyl(1→3)]-6'-methoxy-β-D- glucuronopyranoside (5), and maragenin II 3-O-(β-D-galactopyranosyl(1→2)[β-D-xylopyranosyl(1→2)-β-D-galactopyranosyl(1→3)]-6'-methoxy-β-D-glucuronopyranoside (6), along with two known compounds, (1 and 3), were isolated from the stem bark of Camellia japonica. Their chemical structures were established mainly by 2D NMR techniques and mass spectrometry. The isolated compounds showed inhibitory effects on NO production in RAW264.7 macrophages.     

Switch control pocket inhibitors of p38-MAP kinase. Durable type II inhibitors that do not require binding into the canonical ATP hinge region

Switch control pocket inhibitors of p38-alpha kinase are described. Durable type II inhibitors were designed which bind to arginines (Arg67 or Arg70) that function as key residues for mediating phospho-threonine 180 dependant conformational fluxing of p38-alpha from an inactive type II state to an active type I state. Binding to Arg70 in particular led to potent inhibitors, exemplified by DP-802, which also exhibited high kinase selectivity. Binding to Arg70 obviated the requirement for binding into the ATP Hinge region. X-ray crystallography revealed that DP-802 and analogs induce an enhanced type II conformation upon binding to either the unphosphorylated or the doubly phosphorylated form of p38-alpha kinase.     

Zinc(II) ion mediates tamoxifen-induced autophagy and cell death in MCF-7 breast cancer cell line

Treatment of MCF-7 cells with tamoxifen induced vacuole formation and cell death. Levels of the autophagy marker, microtubule-associated protein light chain 3 (LC3)-II also increased, and GFP-LC3 accumulated in and around vacuoles in MCF-7 cells exposed to tamoxifen, indicating that autophagy is involved in tamoxifen-induced changes. Live-cell confocal microscopy with FluoZin-3 staining and transmission electron microscopy with autometallographic staining revealed that labile zinc(II) ion (Zn²⁺) accumulated in most acidic LC3(+) autophagic vacuoles (AVs). Chelation of Zn²⁺ with N,N,N′,N′-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN) blocked the increase in phospho-Erk and LC3-II levels, and attenuated AV formation and cell death. Conversely, the addition of ZnCl₂ markedly potentiated tamoxifen-induced extracellular signal-regulated kinase (Erk) activation, autophagy and cell death, indicating that Zn²⁺ has an important role in these events. Tamoxifen-induced death was accompanied by increased oxidative stress and lysosomal membrane permeabilization (LMP) represented as release of lysosomal cathepsins into cytosol. Treatment with the antioxidant N-acetyl-l-cysteine (NAC) blunted the increase in Zn²⁺ levels and reduced LC3-II conversion, cathepsin D release and cell death induced by tamoxifen. And cathepsin inhibitors attenuated cell death, indicating that LMP contributes to tamoxifen-induced cell death. Moreover, TPEN blocked tamoxifen-induced cathepsin D release and increase in oxidative stress. The present results indicate that Zn²⁺ contributes to tamoxifen-induced autophagic cell death via increase in oxidative stress and induction of LMP.     

Synthesis,topoisomerase I and II inhibitory activity,cytotoxicity, and structure–activity relationship study of hydroxylated 2,4-diphenyl-6-aryl pyridines

A new series of 2,4-diphenyl-6-aryl pyridines containing hydroxyl group(s) at the ortho, meta, or para position of the phenyl ring were synthesized, and evaluated for topoisomerase I and II inhibitory activity and cytotoxicity against several human cancer cell lines for the development of novel anticancer agents. Structure–activity relationship study revealed that the substitution of hydroxyl group(s) increased topoisomerase I and II inhibitory activity in the order of meta > para > ortho position. Substitution of hydroxyl group on the para position showed better cytotoxicity.     

New antioxidant with dual functions as a peroxidase and chaperone in <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

Thiol-based peroxiredoxins (Prxs) are conserved throughout all kingdoms. We have found that a conserved typical 2-Cys Prx-like protein (PaPrx) from Pseudomonas aeruginosa bacteria displays diversity in its structure and apparent molecular weight (MW), and can act alternatively as a peroxidase and molecular chaperone. We have also identified a regulatory factor involved in this structural and functional switching. Exposure of P. aeruginosa to hydrogen peroxide (H₂O₂) causes PaPrx to convert from a high MW (HMW) complex to a low MW (LMW) form, which triggers a chaperone to peroxidase functional switch. This structural switching is primarily guided by either the thioredoxin (Trx) or glutathione (GSH) systems. Furthermore, comparison of our structural data [native and non-reducing polyacrylamide gel electrophoresis (PAGE) analysis, size exclusion chromatography (SEC) analysis, and electron microscopy (EM) observations] and enzymatic analyses (peroxidase and chaperone assay) revealed that the formation of oligomeric HMW complex structures increased chaperone activity of PaPrx. These results suggest that multimerization of PaPrx complexes promotes chaperone activity, and dissociation of the complexes into LMW species enhances peroxidase activity. Thus, the dual functions of PaPrx are clearly associated with their ability to form distinct protein structures.     

Preventable effect of L-threonate, an ascorbate metabolite, on androgen-driven balding via repression of dihydrotestosterone-induced dickkopf-1 expression in human hair dermal papilla cells

In a previous study, we recently claimed that dihydrotestosterone (DHT)-inducible dickkopf-1 (DKK-1) expression is one of the key factors involved in androgen-potentiated balding. We also demonstrated that L-ascorbic acid 2-phosphate (Asc 2-P) represses DHT-induced DKK-1 expression in cultured dermal papilla cells (DPCs). Here, we investigated whether or not L-threonate could attenuate DHT-induced DKK-1 expression. We observed via RT-PCR analysis and enzyme-linked immunosorbent assay that DHT-induced DKK-1 expression was attenuated in the presence of L-threonate. We also found that DHT-induced activation of DKK-1 promoter activity was significantly repressed by L-threonate. Moreover, a co-culture system featuring outer root sheath (ORS) keratinocytes and DPCs showed that DHT inhibited the growth of ORS cells, which was then significantly reversed by L-threonate. Collectively, these results indicate that L-threonate inhibited DKK-1 expression in DPCs and therefore is a good treatment for the prevention of androgen-driven balding.     

Catalytic properties of a GH10 endo-β-1,4-xylanase from Streptomyces thermocarboxydus HY-15 isolated from the gut of Eisenia fetida

A novel GH10 endo-β-1,4-xylanase (XylG) gene from Streptomyces thermocarboxydus HY-15, which was isolated from the gut of Eisenia fetida, was cloned, over-expressed, and characterized. The XylG gene (1182 bp) encoded a polypeptide of 393 amino acids with a deduced molecular mass of 43,962 Da and a calculated pI of 6.74. The primary structure of XylG was 69% similar to that of Thermobifida fusca YX endo-β-1,4-xylanase. It was most active at pH 6.0 and 55 °C. The susceptibilities of xylans to XylG were as follows: oat spelt xylan > birchwood xylan > beechwood xylan. The XylG also showed high activity (474 IU/mg) toward p-nitrophenylcellobioside. Moreover, at pH 6.0 and 50 °C, the V_max and K_m values of the XylG were 127 IU/mg and 2.51 mg/ml, respectively, for oat spelt xylan and 782 IU/mg and 5.26 mM, respectively, for p-nitrophenylcellobioside. A homology model indicated that XylG folded to form a (β/α)₈-barrel with two catalytic residues of an acid/base (Glu181) and a nucleophile (Glu289). The formation of a disulfide bond between Cys321 and Cys327 were predicted by homology modeling.